RNA sequencing was performed to determine the uniqueness of splenic follicular IgD low B cells compared to splenic follicular IgD high and marginal zone B cells. Overall design: Splenic follicular IgD low and IgD high , and MZ B cells were sorted by FACS from naïve 8-10 weeks old mice. Total RNA was isolated from the sorted cells using RNAqueous® -4PCR kit and RNA sequencing was performed. Splenocytes from five mice were pooled for each sorting. Three independent sorting was performed for each B cell subset.
Mature IgD<sup>low/-</sup> B cells maintain tolerance by promoting regulatory T cell homeostasis.
Specimen part, Cell line, Subject
View SamplesRhoGDIbeta (ARHGDIB) is often expressed in tumor cells. It negatively regulates Rho-GTPases, but may have other functions as well. To analyze its effect on gene expression, RhoGDIbeta was suppressed by RNA interference in MDA-MB-231 breast cancer cells and changes in gene expression monitored by cDNA microarrays.
Cyclooxygenase-2 is a target gene of rho GDP dissociation inhibitor beta in breast cancer cells.
No sample metadata fields
View SamplesOne goal of viral infection is to reprogram the host cell to optimize viral replication. As part of this process, viral miRNAs may compete for components of the miRNA/siRNA pathway as well as regulate cellular targets. Mouse Cytomegalovirus has been described to generate large numbers of viral miRNAs during lytic infection and was therefore used to analyze the impact of viral miRNAs on the host cell small RNA system as well as to check for sorting of viral small RNAs into specific Ago-proteins. Deep sequencing analysis of MCMV infected cells revealed that viral miRNAs represent only app. 13% of all detected miRNAs. All previously described MCMV miRNAs with the exception of miR-m88-1* were confirmed and for the MCMV miR-m01-1 hairpin an additional miRNA, designated miR-m01-1-3p, was found. Its presence was confirmed by qPCR and Northern Blot. Deep sequencing after RISC IP with antibodies specific for either Ago1 or Ago2 showed that all MCMV miRNAs are loaded into both RISC complexes. The ratio of MCMV to mouse miRNAs was not increased after immunoprecipitation of Ago-proteins. Viral miRNAs therefore do not overwhelm the host miRNA processing system nor are they preferentially incorporated into RISC. We found that 3 mouse miRNAs showed an altered expression due to MCMV infection. Down-regulation of miR-27a, as previously described, could be confirmed. In addition, miR-26a was down-regulated and an up-regulation of miR-7a dependent on viral protein expression could be observed. Overall design: Examination of small RNA expression in uninfected vs. infected cells, immunoprecipitation + sequencing of Ago1 and Ago2 loaded small RNAs in infected cells
Murine cytomegalovirus infection of cultured mouse cells induces expression of miR-7a.
Specimen part, Cell line, Subject
View SamplesImmunoprecipitation of EGFR from irradiated A549 (ATCC CCL185) cells was performed in order to characterize bound mRNA species with the help of microarray analysis
New roles for nuclear EGFR in regulating the stability and translation of mRNAs associated with VEGF signaling.
Cell line, Treatment
View SamplesAlternative splicing analysis after treatment with three clinically aproved drugs
Rapid-response splicing reporter screens identify differential regulators of constitutive and alternative splicing.
Cell line, Treatment
View SamplesTherapeutic efficacy of first-generation hypomethylating agents (HMAs) is limited in elderly acute myeloid leukemia (AML) patients. Therefore, combination strategies with targeted therapies are urgently needed. Here, we discover that priming with SGI-110 (guadecitabine), a next-generation HMA, sensitizes AML cells to ASTX660, a novel antagonist of cellular Inhibitor of Apoptosis Protein 1 and 2 (cIAP1/2) and X-linked IAP (XIAP). Importantly, SGI-110 and ASTX660 synergistically induced cell death in a panel of AML cell lines as well as in primary AML samples while largely sparing normal CD34+ human progenitor cells, underlining the translational relevance of this combination. Unbiased transcriptome analysis revealed that SGI-110 alone or in combination with ASTX660 upregulated the expression of key regulators of both extrinsic and intrinsic apoptosis signaling pathways such as TNFRSF10B (DR5), FAS and BAX. Individual knockdown of the death receptors TNFR1, DR5 and FAS significantly reduced SGI-110/ASTX660-mediated cell death, whereas blocking antibodies for TRAIL or FASLG failed to provide protection. Also, TNF-blocking antibody Enbrel had little protective effect on SGI110/ASTX660-induced cell death. Further, SGI-110 and ASTX660 acted in concert to promote cleavage of caspase-8 and BID, thereby providing a link between extrinsic and intrinsic apoptotic pathways. Consistently, sequential treatment with SGI-110 and ASTX660 triggered loss of mitochondrial membrane potential (MMP) and BAX activation, which contributes to cell death as BAX silencing significantly protected from SGI-110/ASTX660-mediated apoptosis. Together, these events culminated in activation of caspases-3/-7, nuclear fragmentation and cell death. In conclusion, SGI-110 and ASTX660 cooperatively induced apoptosis in AML cells by engaging extrinsic and intrinsic apoptosis pathways, highlighting the therapeutic potential of this combination for AML.
Next-generation hypomethylating agent SGI-110 primes acute myeloid leukemia cells to IAP antagonist by activating extrinsic and intrinsic apoptosis pathways.
Cell line
View Sampleswe performed RNA sequencing analysis using 10 tissue samples from human prostate and evaluated efficiency and accuracy of eRNA on mRNA-seq data analysis. Overall design: We sequenced mRNAs from the 10 human tissue samples. After that, we identified mRNAs in these samples against known human genes.
eRNA: a graphic user interface-based tool optimized for large data analysis from high-throughput RNA sequencing.
No sample metadata fields
View SamplesTo gain a deep understanding of mRNA turnover dynamics in mammalian cells, we pulse labeled newly synthesized RNA in 3t3 cells for 2 h with 4sU. RNA samples were fractionated into the newly synthesized and pre-existing fractions. Both fractions and the total RNA sample were analyzed by mRNA sequencing. We estimated mRNA half-lives based on the ratios of newly synthesized RNA/total RNA ratio and the preexisting RNA/total RNA.
Global quantification of mammalian gene expression control.
No sample metadata fields
View SamplesThis project looks into experimentally identifying all minor introns by knocking down the minor spliceosome''s catalytic snRNP, U6atac. Overall design: Knockdown of U6atac by antisense morpholino followed by examining mRNA splicing by RNA-seq
Minor introns are embedded molecular switches regulated by highly unstable U6atac snRNA.
Specimen part, Cell line, Subject
View SamplesWnt signaling pathway is thought to have a role in skin fibrosis in Systemic slcerosis. This Randomized, Placebo-Controlled trial examines the effect of beta catenin inhibition on skin expression.
Inhibition of β-Catenin Signaling in the Skin Rescues Cutaneous Adipogenesis in Systemic Sclerosis: A Randomized, Double-Blind, Placebo-Controlled Trial of C-82.
Treatment, Time
View Samples